Palmitic acid in type 2 diabetes mellitus promotes atherosclerotic plaque vulnerability via macrophage Dll4 signaling

Patients with Type 2 Diabetes Mellitus are increasingly susceptible to atherosclerotic plaque vulnerability, leading to severe cardiovascular events. In this study, we demonstrate that elevated serum levels of palmitic acid, a type of saturated fatty acid, are significantly linked to this enhanced vulnerability in patients with Type 2 Diabetes Mellitus. Through a combination of human cohort studies and animal models, our research identifies a key mechanistic pathway: palmitic acid induces macrophage Delta-like ligand 4 signaling, which in turn triggers senescence in vascular smooth muscle cells. This process is critical for plaque instability due to reduced collagen synthesis and deposition. Importantly, our findings reveal that macrophage-specific knockout of Delta-like ligand 4 in atherosclerotic mice leads to reduced plaque burden and improved stability, highlighting the potential of targeting this pathway. These insights offer a promising direction for developing therapeutic strategies to mitigate cardiovascular risks in patients with Type 2 Diabetes Mellitus.


Figure S1 :
Figure S1: Flowchart of cohort study of "Chronic Stable Coronary Heart

Figure S4 :
Figure S4: Identification of Metabolic Alterations in Human Cohort Serum Samples.OPLS-DA score plots of serum samples analyzed in ESI-(A) and ESI+ (B), respectively.Validation of the OPLS-DA models via 200-times permutation tests in both ESI-(C) and ESI+ (D).The intercepts of R2 and Q2 indicate the robustness of the models and suggest the absence of overfitting.Pairwise comparison results of metabolite concentrations in T2DM versus non-T2DM samples, analyzed in ESI-(E) and ESI+ (F), indicated in volcano plots.Abbreviations: OPLS-DA, orthogonal partial least squares discriminant analysis; ESI-, negative electrospray ionization mode; ESI+, positive electrospray ionization model.

Figure S6 :
Figure S6: Tracking bodyweight and FBG alterations in atherosclerotic mice model complicated with diabetes A. Graphic representation of mouse body weight assessments conducted at 4-week, 8week, and 16-week intervals post STZ or vehicle control injections.B. Chart of FBG measurements recorded at 1-week, 4-week, 6-week, and 16-week periods following STZ or vehicle control injections.Statistical significance is denoted as follows: * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001, with each experimental group comprising 6 mice.Abbreviations: FBG, fasting blood glucose; STZ, streptozotocin

Figure S9 :
Figure S9: Elucidation of Metabolic Variations in Serum Samples of Mice.OPLS-DA score plots of murine serum samples, examined under ESI-(A) and ESI+ (B), respectively.Validation of the robustness and reliability of the OPLS-DA models using permutation tests, performed 200 times for both ESI-(C) and ESI+ (D).The intersecting coordinates of R2 and Q2 ensure the model's strength and negate the risk of overfitting.Volcano plots depicting the pairwise comparison outcomes of metabolite levels in T2DM and non-T2DM samples, assessed under ESI-(E) and ESI+ (F).Abbreviations: OPLS-DA, orthogonal partial least squares discriminant analysis; ESI-, negative electrospray ionization mode; ESI+, positive electrospray ionization model.

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Figure S10: Comparative Metabolomic Analysis in Atherosclerosis with Diabetes (AS+DM) vs. Atherosclerosis-Only (AS) Animals.Metabolomic profiling using KEGG annotation for ESI-(A), ESI+ (B), and COMB (C) between AS+DM and AS animals.Metabolomic characterization leveraging the HMDB annotation in ESI-(D), ESI+ (E), and COMB (F) between AS+DM and AS animals.This comprehensive metabolomic examination underlines the distinct biochemical trajectories observed in atherosclerosis complicated with diabetes (AS+DM) compared to atherosclerosis alone (AS).Abbreviations: AS, Atherosclerosis; DM, Diabetes; KEGG, Kyoto Encyclopedia of Genes and Genomes; HMDB, Human Metabolome Database; COMB, Combined Mode.

Figure S11 :
Figure S11: Integrated Metabolomic Analysis in a Unified Human-Mouse Model.

Figure S14 :
Figure S14: Impact of Palmitic Acid Administration on Dll4 Conditional Knockout Mice in an Atherosclerotic Model This figure examines the physiological and molecular effects of palmitic acid administration in an atherosclerotic mouse model with macrophage-lineage conditional deletion of Dll4 [(Dll4 flox/flox ; Lyz2-Cre +/-; ApoE -/-), henceforth referred to as KO mice], in comparison with their wild-type (WT) ApoE-/-counterparts. Comparative graphical representations illustrating the impact of palmitic acid administration on various physiological parameters in WT and KO mice, including bodyweight (A), fasting blood glucose concentration (B), blood cholesterol

Figure S15 :
Figure S15: Assessment of Dll4 Deletion Specificity and Efficiency in Conditional KO Models A: Confirmation of isolated primary aortic endothelial cells using vwF immunofluorescent staining.Scale bar denotes 40μm.B: Western blot analysis showcasing Dll4 expression in isolated primary aortic endothelial cells from both WT and KO models.C: Identification of isolated primary aortic macrophages via F4/80 immunofluorescent staining.Scale bar is set at 40μm.D: Immunoblot representation of Dll4 levels in primary aortic macrophages isolated from WT and KO samples.E: Validation of isolated primary aortic VSMCs throughα-SMA immunofluorescent staining.Scale bar corresponds to 100μm.F: Western blot depiction of Dll4 expression in isolated primary aortic VSMCs from both WT and KO specimens.Abbreviations: vwF, von Willebrand Factor; VSMCs, Vascular Smooth Muscle Cells; α-SMA:, α-Smooth Muscle Actin.

Figure S16 :
Figure S16: Separate Representative Images of Immunofluorescent Staining of Aortic Root Plaques Presented in this figure are individual, unmerged images from the immunofluorescent staining studies, offering a comprehensive visual supplement to the data provided in Figures 5E and 5F. A. Separate, uncombined images revealing the outcomes of dual immunofluorescent staining for CD68 and Dll4.This method uniquely illuminates the presence of M1 polarized macrophages (signaled by CD68 positivity) and concurrently traces the expression of Dll4 within these immune cells.B. Separate, uncombined images illustrating the outcome of dual immunofluorescent staining for α-SMA and NICD1.In both instances, the counterstaining of cell nuclei with DAPI enhances cellular identification and enumeration.The accompanying scale bar represents a 200μm length.Abbreviations: α-SMA, α-Smooth Muscle Actin; NICD1, Notch Intracellular Domain 1; VSMCs, vascular smooth muscle cells; DAPI, 4',6-diamidino-2-phenylindole

Figure S17 :
Figure S17: Individual CellEvent Green Senescence Staining Images of Aortic Root Plaques This figure showcases distinct, standalone images derived from the CellEvent Green Senescence staining of aortic root plaques, serving as a detailed visual complement to the findings presented in Figures 5F.

Figure S18 :
Figure S18: Effect of Palmitic Acid on M1 Polarization Induction in Primary Macrophages The left panel displays fluorescence microscopy images showcasing CD68 staining (a marker for M1 macrophages), DAPI nuclear staining, and their merged representation for primary macrophages exposed to palmitic acid at concentrations of 0, 0.1, 0.2, and 0.4 mmol/L for a duration of 6 hours.The right panel presents bar graphs depicting the average fluorescence intensity of CD68 across the varying concentrations of palmitic acid treatments.

Figure S19 :
Figure S19: Uncropped Original Western Blot Gels for Targeted Proteins in Figure 6B A. Demonstrates the TLR4 expression in macrophages subjected to palmitic acid incubation and/or Dll4 siRNA transfection.B. Exhibits the levels of phosphorylated ERK in macrophages under similar conditions.C. Depicts total ERK expression in macrophages treated with palmitic acid and/or transfected with Dll4 siRNA.D. Showcases FOXC2 expression in macrophages post palmitic acid incubation and/or Dll4 siRNA transfection.E. Reveals Dll4 expression patterns following palmitic acid treatment and/or Dll4 siRNA transfection.F. Presents GAPDH levels, used as a control, in macrophages exposed to palmitic acid and/or transfected with Dll4 siRNA.Abbreviations: TLR4, toll-like receptor 4; ERK, extracellular signal-regulated kinase; Dll4, delta-like ligand 4; FOXC2, forkhead box C2; GAPDH, glyceraldehyde 3phosphate dehydrogenase